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CHAPTER VI: HARDENING AND ESTABLISHMENT OF THE IN VITRO

RAISED PLANTLETS

INTRODUCTION

The seedlings in culture are continuously exposed to an environment that provides

minimal stress and optimal growth conditions. These seedlings when transferred from the

culture vessels to the community pots require a careful, stepwise procedure which can

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cause the hardening of the seedlings and hence lead to better survival when transplanted

to the pots. Studies have shown that the direct transfer of in vitro raised plantlets to in

vivo environment is restricted (Comer and Thomas 1981; Griffis et ai., 1983). However,

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environmental changes (Brainerd and Fuchiagam, 1981; Roy, 1994; Bamah, 1996).

i The correct method of potting in case of orchids is essential for rapid growth and

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1 development of the plantlets (Kang, 1979). As orchids are delicate plants, they find it

difficult to withstand sudden change of relative humidity during in vitro to in vivo

transfer. Moreover, in vitro raised plantlets fail to withstand direct exposure to harsher

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environment outside the in vitro regimes due to poorly developed cuticle, stomatal

apparatus, photosynthetic ability and conducting tissues (Vij, 1998). Therefore, the first

and foremost requirement for successful transplantation is the maintenance of seedlings

under very high humidity conditions (90-100%) for the first 10-15 days (Bhojwani and

Razdan, 1983) after which gradual reduction of humidity (70-60%) and temperature (28-

38°C) is required (Vij et al., 1995).

Although different types of pots have been used for acclimatization of plantlets,

but the glazed pots are not suitable, as they do not allow sufficient aeration of the roots

and the compost. Mukherjee (1983) suggested the use of clay pots for many epiphytic

orchids like Cattleya, Epidendrum, Dendrobium etc. To facilitate drainage and aeration,

the plastic pots are poked for small holes. According to Hedge (1984), four types of

containers can be used for orchids viz., (i) pots, (ii) baskets or cradles, (iii) wooden logs,

and (iv) tree fern blocks. The use of wooden or bamboo baskets or cradle for epiphytic

orchids has been recommended. However, the use of plastic baskets or copper wire

baskets as containers for orchids is also a common practice.

F or the purpose of hardening, several composts have been proposed and their

effects on growth have been evaluated in different species (Sharma and Chauhan 1995;

Baruah 1996; Sharma and Roy 1996; Sharma and Kaur 1998; Saiprasad and Polisetty;

2003; Kumaria et al. 2005). In the past; the use of fern fibres as suitable compost had

been a common practice (Meyer, 1951). Subsequently, Davidson (1956) proposed a

mixture of coarse peat moss, dried oak leaves, red wood bark fiber (in equal parts) for

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profitable cultivation of epiphytic Cattleya. Brick, charcoal, bark, leaf mould, tree-fern,

dry sphagnum in 1: 1: 1 : 1 : 1:2 ratio proposed by Sharma and Chauhan (1995) showed

100% survivability in Dendrobium chrysanthum. Bark based compost have been used for

the cultivation of Clowesia rosea, Dendrobium alexandrae and Lemboglossum

cervantesii (Stewart, 1988; Cribb, 1990; Robbins and Bell, 1990). Malabadi et af. (2005)

used caorcoal chips, coconut husk and broken tiles (2:2:1) for the compost in case of

Dendrobium nobile. Therefore, an ideal compost which is inert, resistant to organic

decomposition, porous to ensure adequate aeration for root respiration, less costly and

easily available, is mostly supportive for acclimatization of the orchids in the glass house.

Terrestrial orchids like Cymbidium, Paphiopedi/um, Phaius, etc., are generally potted in a

porous media containing loamy soil and adequate organic matter but epiphytic orchids

are held in position by using stakes (Bose and Bhattacharjee, 1980). A majority of

workers have used mixtures of equal parts of chopped tree-fern fiber, chopped sphagnum

moss and crushed bark preparation. Use of fertilizers had been proven to be beneficial for

healthy growth of transferred orchid seedlings. Addition of the nutrients to the compost

varies with the composition of the potting materials and the type of orchid grown.

Apart from container types, compost mixture, fertilizer and humidity,

temperature plays an important critical role in successful transplantation of orchid

plantlets. The best temperature range is reported to be between 18.3°C to 29.4°C. Cribb

(1990) reported 18°C and 23 - 25°C as the minimum and maximum temperatures

respectively for better establishment of Dendrobium alexandrae. In case of

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Phragmipedium bessae, 17°C and 30°C were the required minimum and maximum

suitable temperatures respectively (Robbins, 1989). Similarly, Shiau et al. (2005)

reported the mean day/night temperature of 24°CI18°C for maximum survivability of

Dendrobium candidum plantlets.

The pretreatment of in vitro cultured plantlets before acclimatization to the field

has been useful for successful acclimatization of plantlets. Preconditioning of the

plantlets by growing them in the medium containing high concentrations of sucrose was

reported to influence the in vivo rooting and establishment of cuttings (Wainwright and

Scrace, 1989). Nagaraju and Mani (2005) reported an in vitro pre hardening of

Zygopetelum intermedium in medium containing paclobutrazol and activated charcoal for

its high rate of ex vitro survival and growth of plantlets. Hazarika et al. (2000, 2001) also

reported that in vitro preconditioning of citrus microshoots with sucrose concentrations of

3% was optimum for subsequent ex vitro survival and growth. They also reported that

preconditioning of citrus microshoots with paclobutrazol influences higher ex vitro

survival by intensifying internode length, thickening of root and reducing leaf

dehydration, by regulating the stomatal function and increasing epicuticular wax per unit

area of leaf, besides chlorophyll synthesis.

MATERIALS AND METHODS

In vitro raised complete plantlets of both D. longicornu and D. formosum were

taken for hardening and establishment. Tiny plantlets measuring 2.0-3.0 cm in height

were taken out from the culture tubes/flasks by means of long handled spoon along with a

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small amount of the adhering agar. The agar medium sticking to the roots was removed

slowly with a soft brush and washing with sterile water taking due care to avoid damage

to the roots. The plantlets were then transferred to clean thermocol/plastic pots of 8 cm

diameter containing different mixtures of composts viz., (i) brick pieces and charcoal

chunks (1 : 1), (ii) brick pieces and charcoal chunks (1 : 1) + a top layer of moss, (iii) brick,

charcoal chunks and decaying litter (1:1:1), (iv) brick pieces, charcoal chunks and

decaying litter (1: 1: 1) + a top layer of moss, (v) brick pieces, charcoal and bark pieces

(1: 1: 1), (vi) brick pieces, charcoal chunks and bark pieces (1: 1: 1) + a top layer of moss.

To minimize the spread of disease, thermocol pots were thoroughly washed with

distilled water and dried. The pots were filled with 3/4th compost and watered as planting

in the moistened compost is easier. The washed plantlets were picked up with the help of

forceps and the roots were carefully placed into the crevices of the compost. Single

plantlet was potted in each pot. The pots along with the plantlets were covered with holed

polythene bags for about 2-3 weeks and were carefully sprayed with water and shifted to

the glass house for hardening of the plantlets. The minimum and maximum temperatures

of the glass house at the time of transplantation were 18°C and 25°C respectively. The

relative humidity of the glass house was around 70-80%. The plantlets were watered in

the evening on alternate days and fed with MS nutrient salt solutions (diluted 10 times)

fortnightly for about a month. Readings were recorded after 90 days of hardening and

subsequently the plantlets were transferred to larger earthen pots measuring 25 cm in

diameter.

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RESULTS

Of the various compost combinations used, the compost made up of brick pieces,

charcoal chunks and bark pieces with a layer of moss on top was found to be the most

suitable for the survival of transferred plantlets of D. longicornu wherein 68% survival of

plantlets was recorded (Table 6.1). The compost having brick pieces, charcoal chunks

and decaying litter with a layer of moss on top was also found to be a suitable substratum

for transferred plantlets with a survival of 63%. The compost containing brick pieces,

charcoal chunks without or with a layer of moss did not support good survival of the

transferred plantlets. Also, the other composts used without moss did not support the

survival of the plantlets. In the composts containing brick pieces, charcoal chunks and

decaying litter, brick pieces, charcoal chunks and bark with a layer of moss on top,

plantlets as tall as 4.10 cm and 4.00 cm respectively were obtained. The plantlets were

hardened and established in about 50-60 days time (Plate 6.1 a). Complete established

plantlets were obtained after 90 days. Subsequently, these plantlets were transferred to

earthen pots for further growth and development (Plate 6.1 b).

In case of D. formosum, the composts containing brick pieces, charcoal chunks

and decaying litter with a layer of moss on top was the best for high survivability (87%)

and growth of the in vitro raised plantlets (Table 6.2). The compost containing only brick

pieces and charcoal chunks showed the lowest survivability of the transferred plantlets.

Other substrata used which contained moss as top layers were found to increase the

survival of the transferred plantlets. After 90 days, it was found that the transferred

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Table 6.1: Re-establishment of Dendrobium /ongicornu plantlets after 90

days of hardening

Treatment Survival % Height (em)

Brick + Charcoal (1 : 1 ) 21 ± 1.4 3.44 ± 0.20

Brick + Charcoal (1 : 1 ) 38 ± 2.8 3.55 ± 0.25 + layer of moss

Brick + Charcoal + 32 ± 2.0 3.30 ± 0.11 Decaying litter (1: 1: 1)

Brick + Charcoal 63 ± 4.2 4.10 ± 0.30 +Decaying litter (1: 1: 1) + layer of moss Brick + Charcoal + 35 ± 1.4 3.05 ± 0.25 Bark (1: 1 : 1 )

Brick + Charcoal + 68 ± 2.8 4.00 ± 0.40 Bark (1 : 1 : 1)+ layer of moss

±S.D.

Plate 6.1

Hardened plants of D. longicornu

a. In vitro raised plantlets transferred to plastic pots (after 60 days)

b. Plantlets transferred to earthen pots (after 1 year)

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Table 6.2: Re-establishment of Dendrohium formosum plantlets after 90 days

of hardening

Treatment Survival % Height (em)

Brick + Charcoal (1 : 1) 32 ± 2.8 2.45 ± 0.15

Brick + Charcoal (1: 1) 65 ± 7.0 3.20 ± 0.10 + layer of moss

Brick + Charcoal + 53 ± 4.2 2.55 ± 0.15 Decaying litter (1 : 1 : 1 )

Brick + Charcoal 87 ± 4.2 3.25 ± 0.35 + Decaying litter (1 : 1 : 1) + layer of moss Brick + Charcoal + 37 ± 4.2 2.70 ± 0.10 Bark (1 : 1 : 1)

Brick + Charcoal + 60 ± 2.8 2.55 ± 0.05 Bark (1 : 1 : 1)+ layer of moss

±S.D .

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Plate 6.2

Hardened plants of D. formosum

a. In vitro raised plantlets transferred to thermocol pots (after 90 days)

b. Plantlets after 6 months of transfer

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plantlets of D. formosum were more or less of the same height in all the substrata used.

Feeding the plantlets initially with diluted MS nutrient salt solution for a fortnight proved

to be beneficial for their healthy growth. The plantlets were hardened and established in

40 to 50 days time. Complete established plantlets were developed after 90 days (Plate

6.2a, b), and were subsequently transferred to earthen pots for further growth and

development.

DISCUSSION

The transfer of plantlets from the culture vessels to the glasshouse conditions

requires a careful and stepwise procedure. Successful transplantation also depends on

suitable size of the plantlets and their state of growth in vitro. In the present study,

healthy plantlets showing vigorous growth in the culture vessels were transferred to the

pots. The hardiest and vigorous plants have been found to be easier to transplant as they

are less susceptible to diseases and mechanical injuries. The plantlets transferred to the

pots had healthy and vigorously growing root systems, which ensured better

establishment and growth. The different composts used for both D. longicornu and D.

formosum in this investigation were found to be satisfactory for survivability and normal

growth of the transferred plantlets. The highest survivability of 68% in case of D.

longicornu was obtained on substratum containing brick pieces, charcoal chunks and bark

pieces (1: 1: 1) with a layer of moss on top, while a survivability of 87% in case of

transferred plantlets of D. formosum was obtained on substratum containing brick pieces,

charcoal chunks and decaying litter (1:1 :1) with a top layer of moss. The substrata used

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for both the dendrobes species were found to facilitate proper drainage and aeration for

root respiration. The layer of moss on top proved to be beneficial due to higher retention

of moisture content. It has been reported that successful transplantation requires the

maintenance of seedlings under high humidity for initial periods of transfer (Bhojwai and

Razdan, 1983). The rate of survival and growth of the plantlets of both the species

studied was found to have reduced in a substratum containing only brick and charcoal.

This might be due to the reason that the compost containing only brick pieces and

charcoal chunks could not supply enough nutrients required for the growth of the

transferred plantlets. Also, the compost was too porous and could have leached out the

minimal of the nutrients available. However, the addition of litter to this substratum with

a layer of moss was found to be beneficial for the survival of the transferred plantlets of

both the species. Similar results where decaying litter had been found to be effective on

growth and survivability of plantlets have been reported in Vanda coerulea (Kalita, 1999)

and Dendrobium jimbriatum var. oculatum (Roy and Banerjee, 2003). Baruah (1996)

achieved 90% survivability of plantlets of D. trans parens on potting medium containing

charcoal, brick pieces, chopped fern roots and farmyard manure. In the present study,

bark pieces in the compost were found beneficial for survival and growth of plantlets of

D. longicornu. Sharma and Chauhan (1995) reported 100% survivability of transferred

plantlets of D. chrysanthum on potting media containing brick chips, charcoal, bark

pieces, leaf mould, tree fern and dry sphagnum. The survivability of the micropropagated

plantlets on being transferred to pots depends on proper acclimatization of plantlets. The

best method to ensure optimal survival of cultured plantlets in natural conditions is to

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expose them gradually to a relatively lower humidity, higher temperature and higher light

intensity. Sharma and Chauhan (1995), Vij et al. (1995), and Nagaraju and Mani (2005)

also suggested acclimatization of the in vitro raised plantlets prior to exposure to ex vitro

environment. In the present study, feeding the plantlets with diluted MS nutrient salt

solution was found to be beneficial for growth of the transferred plantlets. This is

consistent with the earlier report of Kumaria and Tandon (1994) wherein in feeding the

plantlets of Dendrobium jimbriatum var. occulatum with diluted MS nutrient salt solution

for the initial period was beneficial as the supply of the essential nutrients to the

transferred plantlets was not stopped abruptly. The promotion of orchid seedling growth

by the nutrienl solution for initial hardening had also been reported earlier in many other

instances (Sander, 1979; Mukherjee, 1983).

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